James D. Foster, Ph.D.
Project: Regulation of Membrane Transporters by Palmitoylation
Mentor: James D. Foster, Ph.D.
Assistant Professor, Department of Biomedical Sciences
Research Project: The dopamine, norepinephrine and serotonin transporters (DAT, NET and SERT, respectively) are membrane proteins responsible for clearance of their corresponding substrates dopamine (DA), norepinephrine (NE) and serotonin (5-HT) from the extraneuronal space during neurotransmission. Each monoamine controls distinct behavioral and physiological functions in the nervous system. Extraneuronal monoamine levels are controlled spatially and temporally by transporter mediated reuptake of released transmitter into presynaptic neurons. Abnormalities in transmitter levels and subsequent neurotransmission are linked to neurological disorders including major depression, schizophrenia, bipolar disorder, attention deficit hyperactivity disorder, Tourette syndrome, and Parkinson disease, through mechanisms that are incompletely understood. In addition, the transporters are sites of action for therapeutic drugs such as methylphenidate, bupropion, selective serotonin and serotonin-norepinephrine reuptake inhibitors, used to treat these disorders, and are also targets for addictive drugs including cocaine, amphetamine (AMPH), and methylenedioxy methamphetamine (MDMA) that elevate transmitter levels.
We previously discovered that rat, mouse and human DATs are modified by S-palmitoylation, a post-translational modification in which C16 saturated palmitic acid is added via a thioester linkage to cysteine. Our studies indicate that DAT palmitoylation has the capacity to impact dopaminergic signaling acutely by regulating DA transport kinetics independent of surface losses and chronically by opposing DAT degradation. We have also identified NET, SERT and the sodium hydrogen ion exchanger isoform 1 (NHE1) as palmitoylated transporters but the sites of modification and their influence on transporter function and membrane microdomain localization are unknown. We have begun to identify the signaling molecules and pathways involved in the regulation of transporter palmitoylation and resulting functional outcomes and our proposed project for the SURF student will be to characterize transporter palmitoylation in response to one of the following: drugs of abuse (AMPH, MDMA, etc), growth factors (insulin, BDNF, etc.) or specific signaling pathway inhibitors, and resulting effects on transport capacity and cell surface transporter expression as time permits.
This project is suitable for execution by an undergraduate student in a 10-week time period since the assays are currently successfully employed in the Foster Lab and conditions used have been worked out for studying DAT palmitoylation. In addition, the studies have scientific merit and relevance to human disease. The study will introduce a student to many pharmacological, biochemical and cell signaling principles related to transporters, drug abuse, and neurologic disorders as well as to basic scientific principles in experimental methodology.
Techniques: The SURF student will learn cell culture with pharmacologic treatments, SDS-PAGE, immunoblotting, acyl-biotinyl exchange (palmitoylation assay), substrate uptake assays with pharmacological principles, cell surface biotinylation assay in addition to statistical analysis, and hypothesis testing. The student will also be given exposure to transporter literature, and the opportunity to write up research findings for posters or papers.
Student Independence: The SURF student will perform daily experiments, with guidance as needed, and will analyze results for discussions with Dr. Foster. Initially, it is expected that more assistance will be needed while the student gains more independence. In the latter weeks of the experience, the student will have significant input into the interpretation of results and planning of further experiments.
